The interaction between virus and antibody ordinarily leads to neutralization, but infectivity of some antibody-coated viruses may be enhanced if susceptible cells bear Fcγ receptors (FcγR). While this apparent paradox has been demonstrated for a number of viruses, it is of particular interest with respect to the dengue viruses. Severe forms of dengue fever, manifested by heightened viremia levels and generalized microvascular leak syndromes (Vaughn et al., “Dengue Viremia Titer, Antibody Response Pattern, and Virus Serotype Correlate with Disease Severity,” J Infect Dis 181(1):2-9 (2000)), have been linked to enhanced infection of monocyte/macrophages by dengue immune complexes (Halstead, S. B., “Antibody, Macrophages, Dengue Virus Infection, Shock, and Hemorrhage: A Pathogenetic Cascade,” Rev Infect Dis 11(Suppl 4):S830-9 (1989); Kliks et al., “Antibody-dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever,” Am J Trop Med Hyg 40(4):444-51 (1989)). Potentially life-threatening forms of dengue fever emerge most often in the course of dengue epidemics when new dengue serotypes (particularly dengue-2) are sequentially introduced into a region. Persuasive seroepidemiologic evidence, clinical cohort studies, and monkey experiments, link pre-existing dengue antibodies, acquired from earlier dengue infection or passively through maternal transmission, to heightened viremia and more severe disease (Halstead, S. B., “Antibody, Macrophages, Dengue Virus Infection, Shock, and Hemorrhage: A Pathogenetic Cascade,” Rev Infect Dis 11(Suppl 4):S830-9 (1989); Halstead, S. B., “Pathogenesis of Dengue: Challenges to Molecular Biology,” Science 239:476-481 (1988); Kliks et al., “Evidence that Maternal Dengue Antibodies are Important in the Development of Dengue Hemorrhagic Fever in Infants,” Am J Trop Med Hyg 38:411-419 (1988); Kliks et al., “Antibody-dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever,” Am J Trop Med Hyg 40(4):444-51 (1989); Vaughn et al., “Dengue Viremia Titer, Antibody Response Pattern, and Virus Serotype Correlate with Disease Severity,” J Infect Dis 181(1):2-9 (2000)).
Several promising multivalent dengue vaccine candidates are in late phases of clinical trial, mainly in dengue-free locales (Halstead & Deen, “The Future of Dengue Vaccines,” Lancet 360:1243-5 (2002)). Their future evaluation in dengue endemic environments poses a unique potential hazard: if the quality of the antibody response to a vaccine component were suboptimal, or if vaccine-stimulated protective antibody levels were to wane, naturally-acquired dengue infection of increased severity might follow.
The nature of enhancing antibodies has been widely investigated using primary monocyte/macrophages or macrophage-like cell lines that express FcγR. Receptor properties that might affect immune enhancement, however, have received comparatively much less attention, largely because heterogeneous FcγR display on such cells complicates interpretation of experimental results.
Enhanced infection of Fc receptor (FcR)-bearing cells of macrophage/monocyte lineage by antibody-complexed dengue virus is central to the pathogenesis of serious forms of dengue fever. Cultured peripheral blood macrophages and macrophage-like cell lines have typically been used to characterize antibodies with respect to enhancing capacity, but results with such cells are confounded by the simultaneous and variable expression of multiple FcR classes and isoforms of differing physiology and by potential ambiguities that arise when antibody and FcR are not of the same species origin. An added problem is failure of dengue virus to form plaques in such cells, so that surrogate amplification methods are needed to measure virus replication. The immune enhancement phenomenon may also be clinically relevant to the pathogenesis of a variety of unrelated RNA and DNA viruses of medical importance.
FcγR comprise a multi-gene family of integral membrane glycoproteins that exhibit complex activation or inhibitory effects on cell functions after aggregation by complexed IgG (Ravetch & Bolland, “IgG Fc Receptors,” Annu Rev Immunol 19:275-90 (2001); Takai, T., “Roles of Fc Receptors in Autoimmunity,” Nat Rev Immunol 2(8):580-92 (2002); Nimmerjahn & Ravetch, “Fcγ Receptors: Old Friends and New Family Members,” Immunity 24:19-28 (2006)). Two activatory human FcγR of different classes and with distinctive, but overlapping, distribution among monocytes known to be permissive to dengue virus infection have been examined. The first, FcγRIA (CD64), is a 72 kD protein found exclusively on antigen-presenting cells of macrophage and dendritic cell lineages, most of which are permissive to dengue virus replication (Fanger et al., “Type I (CD64) and Type II (CD32) Fcγ Receptor-mediated Phagocytosis by Human Blood Dendritic Cells,” J Immunol 157(2):541-8 (1996); Libraty et al., “Human Dendritic Cells are Activated by Dengue Virus Infection: Enhancement by Gamma Interferon and Implications for Disease Pathogenesis,” J Virol 75(8):3501-8 (2001); Wu et al., “Human Skin Langerhans Cells are Targets of Dengue Virus Infection,” Nat Med 6(7):816-20 (2000)). FcγRIA exhibits high affinity for monomeric IgG1 and exists bound to this immunoglobulin in vivo. The second, FcγRIIA (CD32), is a 40 kD protein unique to humans and more broadly distributed among a variety of myelogenous cell types. It has low affinity for monomeric IgG, preferentially binding multivalent IgG (Maenaka et al., “The Human Low Affinity Fcγ Receptors IIa, IIb, and III Bind IgG with Fast Kinetics and Distinct Thermodynamic Properties,” J Biol Chem 276(48):44898-904 (2001)). Each FcγR is comprised of three portions: an extracellular portion of two (FcγRIIA) or three (FcγRIA) IgG-like domains, a short hydrophobic transmembrane region, and a cytoplasmic tail. A conserved immunoreceptor tyrosine-based activation motif (ITAM) links each FcγR to tyrosine kinase-activated signaling pathways that modulate cell metabolism and physical behavior when triggered by receptor clustering (Duchemin et al., “Clustering of the High Affinity Fc Receptor for Immunoglobulin G (FcγRI) Results in Phosphorylation of its Associated γ-Chain,” J Biol Chem 269(16):12111-7 (1994); Letourneur et al., “Characterization of the Family of Dimers Associated with Fc Receptors (FcεRI and FcγRIII),” J Immunol 147(8):2652-6 (1991); Van den Herik-Oudijk et al., “Functional Differences Between Two Fc Receptor ITAM Signaling Motifs,” Blood 86(9):3302-7 (1995); Van den Herik-Oudijk et al., “Functional Analysis of Human FcγRII (CD32) Isoforms Expressed in B Lymphocytes,” J Immunol 152(2):574-85 (1994)). FcγRIA acquires this function by non-covalent association with the γ-chain subunit, a short (ca. 11 kD) transmembrane ITAM-containing homodimer (Kwiatkowska & Sobota, “The Clustered Fcγ Receptor II is Recruited to Lyn-containing Membrane Domains and Undergoes Phosphorylation in a Cholesterol-dependent Manner,” Eur J Immunol 31(4):989-98 (2001)). FcγRIIA, unlike other Fc receptors and most immunoreceptors, incorporates the ITAM in its ligand binding chain.
Signal transduction triggered by ligand engagement is intimately involved in the phagocytosis of IgG opsonized particles where the molecular details of FcγRIA and FcγRIIA signaling have been revealed in exquisite detail (Fitzer-Attas et al., “Fcγ Receptor-mediated Phagocytosis in Macrophages Lacking the Src Family Tyrosine Kinases Hck, Fgr, and Lyn,” J Exp Med 191(4):669-81 (2000); Kim et al., “Fcγ Receptor Transmembrane Domains: Role in Cell Surface Expression, γ Chain Interaction, and Phagocytosis,” Blood 101(11):4479-84 (2003); Kim et al., “Fcγ Receptors Differ in Their Structural Requirements for Interaction with the Tyrosine Kinase Syk in the Initial Steps of Signaling for Phagocytosis,” Clin Immunol 98(1):125-32 (2001); Lowry et al., “Functional Separation of Pseudopod Extension and Particle Internalization During Fcγ Receptor-mediated Phagocytosis,” J Exp Med 187(2):161-76 (1998); Van den Herik-Oudijk et al., “Functional Differences Between Two Fc Receptor ITAM Signaling Motifs,” Blood 86(9):3302-7 (1995)). A signaling requirement for entry of infectious virus immune complexes following FcγR engagement is less certain and has been little studied. One view is that FcγR may facilitate entry of dengue immune complexes by simply concentrating them onto a putative dengue receptor, in essence a passive effect that leads to internalization and infection, perhaps uninfluenced by FcγR signal transduction (Mady et al., “Antibody-dependent Enhancement of Dengue Virus Infection Mediated by Bispecific Antibodies Against Cell Surface Molecules Other Than Fcγ Receptors,” J Immunol 147(9):3139-44 (1991)). Conversely, evidence of differential immune enhancement among FcγR, or for modulation of dengue immune complex infectivity by FcγR-triggered signaling, would have important implications with respect to mechanisms of dengue neutralization and dengue fever pathogenesis.
FcγRIA and FcγRIIA have previously been shown to facilitate antibody-mediated dengue enhancement in human macrophage-like cells using surrogate plaque assays to measure virus replication (Kontny et al., “Gamma Interferon Augments Fcγ Receptor-mediated Dengue Virus Infection of Human Monocytic Cells,” J Virol 62(11):3928-33 (1988); Littaua et al., “Human IgG Fc Receptor II Mediates Antibody-dependent Enhancement of Dengue Virus Infection,” J Immunol 144(8):3183-6 (1990)) since dengue virus does not form plaques in such cells (Peiris & Porterfield, “Antibody-dependent Enhancement of Plaque Formation on Cell Lines of Macrophage Origin—A Sensitive Assay for Antiviral Antibody,” J Gen Virol 57(Pt. 1):119-25 (1981)). A direct assay would help in elucidating the role of various contributors to antibody-dependent enhancement.
A balanced antibody response to multivalent dengue vaccines has not been achieved (Edelman, R., “Dengue and Dengue Vaccines,” JID 191:650-653 (2005); Kitchener et al., “Immunogenicity and Safety of Two Live-attenuated Tetravalent Dengue Vaccine Formulations in Healthy Australian Adults,” Vaccine 24(9):1238-41 (2006); Sun et al., “Protection of Rhesus Monkeys Against Dengue Virus Challenge after Tetravalent Live Attenuated Dengue Virus Vaccination,” JID 193:1658-1665 (2006); Edelman et al., “Phase I Trial of 16 Formulations of a Tetravalent Live-attenuated Dengue Vaccine,” Am J Trop Med Hyg 69(Suppl 6):48-60 (2003); Blaney, Jr. et al., “Recombinant, Live-attenuated Tetravalent Dengue Virus Vaccine Formulations Induce a Balanced, Broad, and Protective Neutralizing Antibody Response Against Each of the Four Serotypes in Rhesus Monkeys,” J Virol 79(9):5516-5528 (2005); Guirakhoo et al., “Viremia and Immunogenicity in Nonhuman Primates of a Tetravalent Yellow Fever-Dengue Chimeric Vaccine Genetic Reconstructions, Dose Adjustment, and Antibody Responses against Wild-type Dengue Virus Isolates,” Virol 298:146-159 (2002)). An assay that could be used to screen for neutralization and/or enhancement by candidate vaccines would be helpful in vaccine development.
The present invention is directed to overcoming these and other deficiencies in the art.